U.S. patent number 5,992,226 [Application Number 09/074,541] was granted by the patent office on 1999-11-30 for apparatus and method for measuring intermolecular interactions by atomic force microscopy.
This patent grant is currently assigned to The United States of America as represented by the Secretary of the Navy. Invention is credited to John-Bruce DeVault Green, Gil U Lee.
United States Patent |
5,992,226 |
Green , et al. |
November 30, 1999 |
Apparatus and method for measuring intermolecular interactions by
atomic force microscopy
Abstract
A sample support member for atomic force microscopy of
intermolecular interactions includes a sample support base having a
plurality of protrusions, each protrusion having an apical
substrate region or tip that has been chemically modified by the
immobilization thereon of a sample compound or of a linking
compound that is capable of binding a sample compound. A reference
compound support member has a surface region having at least one
reference compound immobilized thereon. The relative position and
orientation of the reference compound support member and the
substrate support member are controlled to select a particular
protrusion and to cause an interaction between a reference compound
immobilized on the surface region of the free end of the cantilever
and the sample compound immobilized on the apical substrate area of
the selected protrusion. A physical parameter associated with the
interaction between the reference compound and the sample compound
can be measured.
Inventors: |
Green; John-Bruce DeVault
(Alexandria, VA), Lee; Gil U (Alexandria, VA) |
Assignee: |
The United States of America as
represented by the Secretary of the Navy (Washington,
DC)
|
Family
ID: |
22120112 |
Appl.
No.: |
09/074,541 |
Filed: |
May 8, 1998 |
Current U.S.
Class: |
73/105; 977/849;
977/863; 977/851; 977/853; 850/33; 850/9 |
Current CPC
Class: |
B82Y
35/00 (20130101); G01Q 60/42 (20130101); Y10S
977/863 (20130101); Y10S 977/849 (20130101); Y10S
977/851 (20130101); Y10S 977/853 (20130101) |
Current International
Class: |
G12B
21/08 (20060101); G12B 21/00 (20060101); G12B
21/20 (20060101); G01N 027/00 () |
Field of
Search: |
;73/105
;250/306,307 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Erlandsson et al., "Scanning Force Microscopy-Examples of
Application to face Chemistry", received Jun. 1992, pp. 1-21. .
Nakagawa et al., Discriminating Molecular Length of Chemically
Absorbed Molecules Using an Atomic Force Microscope Having a Tip
Covered With Sensor Molecules (An Atomic Force Microscope Having
Chemical Sensing Function), Jpn. J. Appl. Phys., vol. 32, Par.
.
Florin E.-L. et al., "Adhesion Forces Between Individual
Ligand-Receptor Pairs" Science 264 (1994) pp. 415-417. .
Lee et al., "Sensing Discrete Streptavidin-Biotin Interactions with
Atomic Force Microscopy" Langmuir, vol. 10 (2), (1994) pp. 354-357.
.
Dammer et al., "Specific Antigen/Antibody Interactions Measured by
Force Microscopy" Biophysical Journal vol. 70 (May 1996) pp.
2437-2441. .
Chilikoti et al., "The Relationship Between Ligand-Binding
Thermodynamics and Protein-Ligand Interaction Forces Measured by
Atomic Force Microscopy" Biophysical Journal vol. 69 (Nov. 1995)
pp. 2125-2130. .
Allen S. et al., "Detection of Antigen-Antibody Binding Events with
the Atomic Force Microscope" Biochemistry, vol. 36, No. 24 (1997)
pp. 7457-7463. .
Moy V.T. et al., "Adhesive Forces Between Ligand and Receptor
Measured by AFM" Colloids and Sufaces A: Physicochemical and
Engineering Aspects 93 (1994) pp. 343-348. .
Prater, C.B. et al., "Probing Nano-Scale Forces With The AFM"
Digital Instruments, Application Notes, at
http://www.di.com/AppNotes/ForcCurv/FCMain.html (1995). .
"Ultrasharp Silicon Gratings" NT-MDT web site at
http://www.ntmdt.ru/grating.htm and
http://www.ntmdt.ru/manual/silgrat.htm ..
|
Primary Examiner: Larkin; Daniel S.
Attorney, Agent or Firm: Edelberg; Barry A. Webb; Ralph
T.
Claims
What is claimed is:
1. A sample support member for atomic force microscopy comprising a
sample support base having a plurality of tapered or rounded
protrusions, each protrusion having an apical substrate region that
has been chemically modified by the immobilization thereon of a
sample compound or of a linking compound that is capable of binding
a sample compound, wherein the sample compounds or linking
compounds on each of the protrusions may be the same or
different.
2. The sample support member of claim 1 wherein the protrusions are
pyramidal.
3. The sample support member of claim 1 wherein the protrusions are
conical.
4. The sample support member of claim 1 wherein the protrusions are
hemispherical.
5. The sample support member of claim 1 wherein the height of each
protrusions is between about 1 and about 10 .mu.m, the apical
radius of curvature of each protrusions is between about 5 and
about 1000 nm, and the distance between adjacent protrusions is
between about 2 and about 50 .mu.m.
6. The sample support member of claim 5 wherein each protrusion has
a radius of curvature sufficiently small so that a single isolated
molecule of a sample compound can be immobilized on the apical
substrate region.
7. The sample support member of claim 1 wherein the apical
substrate regions of the protrusions have been chemically modified
by the immobilization thereon of sample compounds that are members
of a combinatorial library.
8. The sample support member of claim 7 wherein the sample
compounds have been synthesized in situ on the apical substrate
regions of the protrusions.
9. The sample support member of claim 1 wherein the protrusions are
in a planar array.
10. The sample support member of claim 1 wherein the sample support
member is in the form of a plurality of terraces, each terrace
having at least one row of protrusions.
11. The sample support member of claim 1 wherein the sample support
base is coupled to a force transducer.
12. The sample support member of claim 1 wherein the apical
substrate regions of the protrusions have been chemically modified
by the immobilization thereon of a linking compound that is capable
of binding a thiol group of a thiol-containing sample compound.
13. The sample support member of claim 1 wherein the apical
substrate regions of the protrusions have been chemically modified
by the immobilization thereon of a linking compound that is capable
of binding an amine group of an amine-containing compound.
14. The sample support member of claim 1 wherein the apical
substrate regions of the protrusions have been chemically modified
by the immobilization thereon of a linking compound that is
photoactivatable.
15. The sample support member of claim 1, further including means
for distinguishing the protrusions from each other by optical
microscopy.
16. The sample support member of claim 15 wherein the means for
distinguishing the protrusions from each other comprises markings
on the sample support base that are visible through an optical
microscope.
17. A sample support member for atomic force microscopy comprising
a sample support base having a planar array of tapered or rounded
protrusions, wherein the height of each protrusions is between
about 1 and about 10 .mu.m, the apical radius of curvature of each
protrusions is between about 5 and about 1000 nm, and the distance
between adjacent protrusions is between about 2 and about 50 .mu.m
and wherein each protrusion has an apical substrate region that has
been chemically modified by the immobilization thereon of a sample
compound or of a linking compound that is capable of binding a
sample compound, wherein the sample compounds or linking compounds
on each of the protrusions may be the same or different.
18. A cantilever for an atomic force microscope comprising a
cantilever body having a fixed end and a free end, the free end
having a surface region having a plurality of spatially addressable
subregions, each subregion having been chemically modified by the
immobilization of a different reference compound thereon.
19. The cantilever of claim 18 wherein the surface region of the
free end of the cantilever is substantially flat and has a surface
area of at least 0.01 square microns.
20. A cantilever for an atomic force microscope comprising a
cantilever body having a fixed end and a free end, the free end
having a surface region, the surface region having a plurality of
beads of different size categories immobilized thereon, the beads
having reference compounds immobilized thereon, wherein each size
category of bead has a different reference compound immobilized
thereon.
21. An apparatus for measuring an interaction between a reference
compound and at least one sample compound, the apparatus
comprising:
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region that has been
chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different,
a reference compound support member having a surface region that
has been chemically modified by the immobilization of at least one
reference compound thereon,
means for controlling the relative position and orientation of the
reference compound support member and the sample support member to
select a particular protrusion and to cause an interaction between
the reference compound immobilized on the surface region of the
reference compound support member and the sample compound
immobilized on the apical substrate area of the selected
protrusion, and
means for measuring a physical parameter associated with the
interaction between the reference compound and the sample
compound.
22. The apparatus of claim 21 wherein the protrusions are tapered
or rounded.
23. The apparatus of claim 21 wherein the protrusions are
pyramidal.
24. The apparatus of claim 21 wherein the protrusions are
conical.
25. The apparatus of claim 21 wherein the protrusions are
hemispherical.
26. The apparatus of claim 21 wherein the protrusions are in a
planar array.
27. The apparatus of claim 21 wherein the height of each
protrusions is between about 1 and about 10 .mu.m, the apical
radius of curvature of each protrusions is between about 5 and
about 1000 nm, and the distance between adjacent protrusions is
between about 2 and about 50 .mu.m.
28. The apparatus of claim 21 wherein the chemically modified
surface region of the reference compound support member is
substantially flat and has a surface area of at least 0.01 square
microns.
29. The apparatus of claim 21 wherein the chemically modified
surface region of the reference compound support member includes a
plurality of spatially addressable subregions, each subregion
having been chemically modified by the immobilization of a
different reference compound thereon.
30. The apparatus of claim 21 wherein the surface area of the
apical substrate region of each protrusion is less than the surface
area of the surface region of the free end of the cantilever.
31. The apparatus of claim 21 wherein the reference compound is a
receptor and the sample compounds are known or candidate
complimentary ligands.
32. The apparatus of claim 21 wherein the reference compound is a
ligand and the sample compounds are known or candidate
complimentary receptors.
33. The apparatus of claim 21 wherein the reference compound is an
antibody and the sample compounds are known or candidate
complimentary antigens.
34. The apparatus of claim 21 wherein the reference compound is an
antigen and the sample compounds are known or candidate
complimentary antibodies.
35. The apparatus of claim 21 wherein the reference compound is a
single strand of DNA or RNA and the sample compounds are known or
candidate complimentary strands of DNA or RNA.
36. The apparatus of claim 21 wherein the sample compounds are
members of a combinatorial library.
37. The apparatus of claim 21 wherein the sample compounds are
members of a combinatorial library and wherein each sample compound
has been synthesized in situ on the apical substrate region of a
protrusion of the sample support member.
38. The apparatus of claim 21, wherein the reference compound
support member and the sample support member are immersed in a
fluid.
39. The apparatus of claim 21 wherein the means for measuring a
physical parameter associated with the interaction between the
reference compound and the sample compound comprises a force
transducer coupled to the sample support member.
40. The apparatus of claim 21 wherein the means for measuring a
physical parameter associated with the interaction between the
reference compound and the sample compound comprises a force
transducer coupled to the reference compound support member.
41. The apparatus of claim 21 wherein the reference compound
support member is a cantilever having a fixed end and a free end,
and wherein the chemically modified surface region is on the free
end of the cantilever.
42. An apparatus for measuring an interaction between at least one
reference compound and at least one sample compound by atomic force
microscopy, the apparatus comprising:
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region that has been
chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different,
a cantilever having a fixed end and a free end, the free end having
a surface region that has been chemically modified by the
immobilization of at least one reference compound thereon,
means for controlling the relative position and orientation of the
cantilever and the sample support member to select a particular
protrusion and to cause an interaction between a reference compound
immobilized on the surface region of the free end of the cantilever
and the sample compound immobilized on the apical substrate area of
the selected protrusion, and
means for measuring a physical parameter associated with the
interaction between the reference compound and the sample
compound.
43. The apparatus of claim 42 wherein the chemically modified
surface region of the cantilever includes a plurality of spatially
addressable subregions, each subregion having been chemically
modified by the immobilization of a different reference compound
thereon.
44. An apparatus for measuring an interaction between at least one
reference compound and at least one sample compound by atomic force
microscopy, the apparatus comprising:
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region that has been
chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different,
a cantilever having a fixed end and a free end, the free end having
a surface region that has been chemically modified by the
immobilization of at least one reference compound thereon,
wherein the sample support member is adapted to interface with a
sample stage portion of an atomic force microscope and the
cantilever is adapted to interface with cantilever-holding portion
of an atomic force microscope.
45. An apparatus for measuring an interaction between at least one
reference compound and at least one sample compound by atomic force
microscopy, the apparatus comprising:
a sample support member having a planar array of tapered or rounded
protrusions, wherein the height of each protrusions is between
about 1 and about 10 .mu.m, the apical radius of curvature of each
protrusions is between about 5 and about 1000 nm, and the distance
between adjacent protrusions is between about 2 and about 50 .mu.m
and wherein each protrusion has an apical substrate region that has
been chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different,
a cantilever having a fixed end and a free end, the free end having
a substantially flat surface region that has been chemically
modified by the immobilization of at least one reference compound
thereon, the surface region having an area of at least 0.01 square
microns,
wherein the sample support member is adapted to interface with a
sample stage portion of an atomic force microscope and the
cantilever is adapted to interface with a cantilever-holding
portion of an atomic force microscope.
46. The apparatus of claim 45 wherein the chemically modified
surface region of the cantilever includes a plurality of spatially
addressable subregions, each subregion having been chemically
modified by the immobilization of a different reference compound
thereon.
47. A method of assaying at least one sample compound for
interaction with at least one reference compound, the method
comprising: the steps of
(a) providing (1) a sample support member having a plurality of
protrusions, each protrusion having an apical substrate region and
(2) a reference compound support member having a surface
region,
(b) chemically modifying the apical substrate region of each
protrusion of the sample support member to immobilize a sample
compound thereon, wherein the sample compounds on each of the
protrusions may be the same or different,
(c) chemically modifying the surface region of the reference
compound support member to immobilize at least one reference
compound thereon,
(d) coupling the sample support member and the reference compound
support member to apparatus that includes means for controlling the
relative position and orientation of the sample support member and
the reference compound support member to select a particular
protrusion of the sample support member and to cause an interaction
between a reference compound immobilized on the surface region of
the reference compound support member and the sample compound
immobilized on the apical substrate area of the selected
protrusion, and means for measuring a physical parameter associated
with the interaction between the reference compound and the sample
compound
(e) controlling the relative position and orientation of the
reference compound support member and the sample support member to
select a particular protrusion and to cause an interaction between
a reference compound and the sample compound immobilized on the
selected protrusion, and
(f) measuring a physical parameter associated with the interaction
between the reference compound and the sample compound on the
selected protrusion.
48. The method of claim 47 wherein, at the conclusion of step (f),
steps (e) and (f) are repeated with a different selected protrusion
and/or a different reference compound.
49. A method of assaying at least one sample compounds for
interaction with at least one reference compound, the method
comprising: the steps of
(a) providing an atomic force microscope that includes
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region and
a cantilever having a fixed end and a free end, the free end having
a surface region having a plurality of spatially addressable
subregions,
(b) chemically modifying the apical substrate region of each
protrusion so that each protrusion has the same or a different
sample compound immobilized thereon,
(c) chemically modifying the spatially addressable subregions of
the surface region of the free end of the cantilever to immobilize
a different reference compound on each subregion,
(d) controlling the relative position and orientation of the
cantilever and the sample support member to select a particular
subregion of the cantilever and a particular protrusion to cause an
interaction between the reference compound immobilized on the
selected subregion and the sample compound immobilized on the
selected protrusion, and
(e) measuring a physical parameter associated with the interaction
between the selected reference compound and the selected sample
compound.
50. A method of assaying at least one sample compounds for
interaction with at least one reference compound, the method
comprising: the steps of
(a) providing an apparatus that includes
a sample support member having a planar array of tapered or rounded
protrusions, wherein the height of each protrusions is between
about 1 and about 10 .mu.m, the apical radius of curvature of each
protrusions is between about 5 and about 1000 nm, and the distance
between adjacent protrusions is between about 2 and about 50 .mu.m
and wherein each protrusion has an apical substrate region that has
been chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different, and
a cantilever having a fixed end and a free end, the free end having
a substantially flat surface region having an area of at least 0.01
square microns and wherein the surface region is subdivided into a
plurality of spatially addressable subregions,
(b) chemically modifying the apical substrate region of each
protrusion to immobilize a sample compound thereon, wherein the
sample compounds on each of the protrusions may be the same or
different,
(c) chemically modifying the spatially addressable subregions of
the surface region of the free end of the cantilever to immobilize
a different reference compound on each subregion,
(d) coupling the sample support member to a sample stage portion of
an atomic force microscope and coupling the cantilever to a
cantilever-holding portion of the atomic force microscope, wherein
the atomic force microscope includes means for controlling the
relative position and orientation of the cantilever and the sample
support member to select a particular protrusion and to cause an
interaction between a reference compound immobilized on the surface
region of the free end of the cantilever and the sample compound
immobilized on the apical substrate area of the selected
protrusion, and means for measuring a physical parameter associated
with the interaction between the reference compound and the sample
compound,
(e) controlling the relative position and orientation of the
cantilever and the sample support member to select a particular
protrusion and to cause an interaction between a reference compound
immobilized on the selected subregion and the sample compound
immobilized on the selected protrusion, and
(f) measuring a physical parameter associated with the interaction
between the selected reference compound and the selected sample
compound.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to atomic force microscopy
(AFM) and to apparatus and methods for measuring intermolecular
interactions such as receptor/ligand, antibody/antigen and DNA
interactions efficiently by atomic force microscopy.
2. Description of the Related Art
In conventional atomic force microscopy, a sample is scanned with a
fine-tipped probe mounted on a cantilever, and deflections of the
probe tip as it passes across the sample are measured to determine
the topography of the sample. Deflections in the probe tip as it
passes along the sample surface may be monitored by various
methods, including optical reflection, interferometry, and
piezoelectric strain gauge methods. See, for example, U.S. Pat. No.
Re. 33,387 to Binnig, U.S. Pat. No. 5,144,833 to Amer et al, U.S.
Pat. No. 5,463,897 to Prater et al, U.S. Pat. No. Re. 34,489 to
Hansma et al and U.S. Pat. No. 5,260,824 to Okada et al, all of the
above incorporated herein by reference.
In recent years, atomic force microscopy has been used to measure
interfacial properties and intermolecular interactions such as
elasticity, friction, adhesion, receptor/ligand interactions, and
antibody/antigen interactions between individual molecules. To
measure binding interactions between complimentary ligands and
receptors, the probe tip of a cantilever and a sample surface may
be chemically modified by various means to attach the complementary
ligands and receptors to each. (For convenience, the compound or
compounds immobilized on the cantilever or similar structures will
be referred to herein as "reference" compounds and the compound or
compounds immobilized on the sample support member or sample
surface will be referred to herein as "sample" compounds.) The
probe tip and sample surface may then be brought into proximity or
into contact so that a ligand and a receptor interact or bond. When
the probe tip and sample surface are then separated, the
ligand-receptor bond breaks and the strength of the interaction
force between the ligand and the receptor may be measured. The use
of atomic force microscopy to study intermolecular forces is
described, for example, in the following patents and publications,
incorporated herein by reference: U.S. Pat. No. 5,363,697 to
Nakagawa; U.S. Pat. No. 5,372,930 to Colton et al; Florin E.-L. et
al, "Adhesion Forces Between Individual Ligand-Receptor Pairs"
Science 264 (1994). pp 415-417; Lee, G.U et al, "Sensing Discrete
Streptavidin-Biotin Interactions with Atomic Force Microscopy"
Langmuir, vol. 10(2), (1994) pp 354-357; Dammer U. et al "Specific
Antigen/Antibody Interactions Measured by Force Microscopy"
Biophysical Journal Vol. 70 (May 1996) pp 2437-2441; Chilikoti A.
et al, "The Relationship Between Ligand-Binding Thermodynamics and
Protein-Ligand Interaction Forces Measured by Atomic Force
Microscopy" Biophysical Journal Vol. 69 (November 1995) pp
2125-2130; Allen S. et al, "Detection of Antigen-Antibody Binding
Events with the Atomic Force Microscope" Biochemistry, Vol. 36, No.
24 (1997) pp 7457-7463; and Moy V.T. et al, "Adhesive Forces
Between Ligand and Receptor Measured by AFM" Colloids and Surfaces
A: Physicochemical and Engineering Aspects 93 (1994) pp 343-348. If
interactions between molecules are studied in liquids, the
experimental conditions, such as pH, buffer/ionic concentration,
buffer/ionic species, etc. may be varied to determine the effect
that these have on the forces of interaction.
Atomic force microscopy has great potential for use in screening
arrays of compounds, such as libraries of compounds produced by
combinatorial methods, to identify useful ligand/receptor
interactions and to discover useful drugs. Modem methods of
combinatorial chemistry, parallel synthesis, and microlithography
make it possible to produce large, compact libraries of chemical
analogues in spatially addressable arrays. Atomic force microscopy
with chemically modified probe tips provides a way of screening
these arrays. However, the equipment and techniques currently used
for atomic force microscopy are not well suited for repetitive
measurements or efficient high-volume screening. In particular,
chemically modified cantilever probe tips are fragile and easily
damaged or inactivated. In a typical chemically modified cantilever
probe tip, only the molecules that are bound to the very apex of
the tip are available for force interactions with a substrate. The
crucial area of the probe tip is typically very small and the
number of molecules bound thereon is very few; if anything happens
to damage those few molecules or to block access to, or to
otherwise inactivate that small area of the probe tip, then the
probe tip is rendered useless and must be replaced. Replacing the
probe tip usually requires replacing the entire cantilever, a
procedure that is expensive and time-consuming. Moreover, the
typical cantilever probe tip has room for only one reference
compound to be immobilized on the tip, so the cantilever must be
replaced or modified whenever it is desired to use a different
reference compound.
SUMMARY OF THE INVENTION
It has now been discovered that the limitations of atomic force
microscopy for use in measuring intermolecular interactions as
described above may be overcome by making two concurrent changes to
the atomic force microscope apparatus. First, the sample support is
modified so that instead of being a flat surface having molecules
of the sample compound or compounds spread out over a relatively
large area, it has a plurality of protrusions having molecules of
the sample compound or compounds immobilized on the tips or apices
of the protrusions. The sample support member of this invention may
contain millions of these protrusions per square centimeter.
Immobilizing the sample compounds onto the tips of protrusions
instead of onto a flat surface places the sample compounds in more
accessible position so that when one conducts measurements of
molecular interactions of a sample compound with a reference
compound, it is not necessary to put the reference compound on the
end of a cantilever probe tip to access the sample molecules.
Individual molecules of the sample compounds can be accessed by
reference compounds that are immobilized on a surface on the
cantilever. Thus, the second change to the atomic force apparatus
is to modify the cantilever so that the reference compound or
compounds are immobilized directly onto a surface region on the
free end of the cantilever instead of on a probe tip.
In the operation of the atomic force microscope to measure
molecular interactions according to the present invention, the
surface region of the free end of the cantilever is brought into
contact with or into proximity with a particular protrusion so that
interactions between a molecule of the reference compound and a
molecule of the sample compound can be measured. Piezoelectric
translators currently used in atomic force microscopy are
sufficiently precise so that a particular protrusion out of
millions on the sample support member can be aligned with a
particular portion of the surface region of the cantilever.
The protrusions in the present invention are analogous to the
cantilever probe tip in conventional atomic force microscopy in
that they allow a small number of individual molecules to be
isolated in a small area in an accessible geometric configuration
so that measurements on single molecules or on a small number of
molecules may be taken. A major advantage of the present invention
is that, whereas in conventional atomic force microscopy, there is
only one probe tip, which located on the cantilever, in the present
invention, there are numerous analogous structures, the
protrusions, which are located on the sample support member. In a
conventional atomic force microscope, if the cantilever probe tip
is damaged or inactivated, an experiment must be halted until the
tip is replaced. In the present invention, if a particular
protrusion is damaged or inactivated, one may simply reposition the
cantilever over a different protrusion having the same sample
compound immobilized thereon and continue with the process of
taking measurements. Thousands or millions of protrusions per
square centimeter can be created and chemically modified with a
sample compound or compounds.
The surface region of the cantilever can have billions or trillions
of molecules of the reference compound immobilized on it, each of
which is usable in the measurement of interactions with a sample
compound immobilized on a protrusion. If molecules of the reference
compound located on one part of the cantilever become damaged or
inactivated in the course of an experiment, the cantilever may be
repositioned so that undamaged, active molecules of the reference
compound located on a different portion of the cantilever can be
used instead. Moreover, the surface region of the cantilever can be
subdivided into spatially addressable subregions each having a
different reference compound immobilized thereon. A plurality of
different reference compounds can be immobilized on discrete,
spatially addressable subregions of the surface of the cantilever,
so there is no need to replace the cantilever every time it is
desired to use a different reference compound.
By making the changes to the sample support and to the cantilever
as described above, it is now possible to use atomic force
microscopy for efficient, high-volume screening of sample compounds
and for taking repetitive measurements of molecular interactions so
that results may be double-checked with statistical rigor without
the researcher having to worry about needing to replace the
cantilever in the middle of an experiment because of damage to or
inactivation of the reference compound.
The apparatus and methods of the present invention are not limited
to use with cantilever-based atomic force microscopy instruments.
The sample support member may also be used to hold sample compounds
for other types of scanning microscopy in addition to those that
involve the use of a cantilever. In general terms, the sample
compound or sample compounds are immobilized on the apical
substrate regions of protrusions of a sample support member, and
the reference compound or reference compounds are immobilized on
the surface region of a reference compound support member. Any
method known in the art may be used to control the relative
position of the sample support member and the reference compound
support member and to measure intermolecular interactions between
sample compounds and reference compounds.
Accordingly, in one aspect, the invention provides a sample support
member for atomic force microscopy comprising a sample support base
having a plurality of protrusions, each protrusion having an apical
substrate region that has been chemically modified by the
immobilization thereon of a sample compound or of a linking
compound that is capable of binding a sample compound, wherein the
sample compounds or linking compounds on each of the protrusions
may be the same or different.
In another aspect, the invention provides a cantilever for an
atomic force microscope comprising a cantilever body having a fixed
end and a free end, the free end having a surface region having a
plurality of spatially addressable subregions, each subregion
having been chemically modified by the immobilization of a
different reference compound thereon.
In another aspect, the invention provides a cantilever for an
atomic force microscope comprising a cantilever body having a fixed
end and a free end, the free end having a surface region, the
surface region having a plurality of beads of different size
categories immobilized thereon, the beads having reference
compounds immobilized thereon, wherein each size category of bead
has a different reference compound immobilized thereon.
In another aspect, the invention provides an apparatus for
measuring an interaction between a reference compound and at least
one sample compound, the apparatus comprising
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region that has been
chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different,
a reference compound support member having a surface region that
has been chemically modified by the immobilization of at least one
reference compound thereon,
means for controlling the relative position and orientation of the
reference compound support member and the sample support member to
select a particular protrusion and to cause an interaction between
the reference compound immobilized on the surface region of the
reference compound support member and the sample compound
immobilized on the apical substrate area of the selected
protrusion, and
means for measuring a physical parameter associated with the
interaction between the reference compound and the sample
compound.
In another aspect, the invention provides an apparatus for
measuring an interaction between at least one reference compound
and at least one sample compound by atomic force microscopy, the
apparatus comprising
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region that has been
chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different,
a cantilever having a fixed end and a free end, the free end having
a surface region that has been chemically modified by the
immobilization of at least one reference compound thereon,
wherein the sample support member is adapted to interface with a
sample stage portion of an atomic force microscope and the
cantilever is adapted to interface with a cantilever-holding
portion of an atomic force microscope.
In another aspect, the invention provides a method of assaying at
least one sample compound for interaction with at least one
reference compound, the method comprising the steps of
(a) providing (1) a sample support member having a plurality of
protrusions, each protrusion having an apical substrate region and
(2) a reference compound support member having a surface
region,
(b) chemically modifying the apical substrate region of each
protrusion of the sample support member to immobilize a sample
compound thereon, wherein the sample compounds on each of the
protrusions may be the same or different,
(c) chemically modifying the surface region of the reference
compound support member to immobilize at least one reference
compound thereon,
(d) coupling the sample support member and the reference compound
support member to apparatus that includes means for controlling the
relative position and orientation of the sample support member and
the reference compound support member to select a particular
protrusion of the sample support member and to cause an interaction
between a reference compound immobilized on the surface region of
the reference compound support member and the sample compound
immobilized on the apical substrate area of the selected
protrusion, and means for measuring a physical parameter associated
with the interaction between the reference compound and the sample
compound
(e) controlling the relative position and orientation of the
reference compound support member and the sample support member to
select a particular protrusion and to cause an interaction between
a reference compound and the sample compound immobilized on the
selected protrusion, and
(f) measuring a physical parameter associated with the interaction
between the reference compound and the sample compound on the
selected protrusion.
In another aspect, the invention provides a method of assaying at
least one sample compound for interaction with at least one
reference compound, the method comprising the steps of
(a) providing an atomic force microscope that includes
a sample support member having a plurality of protrusions, each
protrusion having an apical substrate region and
a cantilever having a fixed end and a free end, the free end having
a surface region having a plurality of spatially addressable
subregions,
(b) chemically modifying the apical substrate region of each
protrusion so that each protrusion has the same or a different
sample compound immobilized thereon,
(c) chemically modifying the spatially addressable subregions of
the surface region of the free end of the cantilever to immobilize
a different reference compound on each subregion,
(d) controlling the relative position and orientation of the
cantilever and the sample support member to select a particular
subregion of the cantilever and a particular protrusion to cause an
interaction between the reference compound immobilized on the
selected subregion and the sample compound immobilized on the
selected protrusion, and
(e) measuring a physical parameter associated with the interaction
between the selected reference compound and the selected sample
compound.
In another aspect, the present invention provides a method of
assaying at least one sample compound for interaction with at least
one reference compound, the method comprising the steps of
(a) providing an apparatus that includes
a sample support member having a planar array of tapered or rounded
protrusions, wherein the height of each protrusions is between
about 1 and about 10 .mu.m, the apical radius of curvature of each
protrusions is between about 5 and about 1000 nm, and the distance
between adjacent protrusions is between about 2 and about 50 .mu.m
and wherein each protrusion has an apical substrate region that has
been chemically modified by the immobilization of a sample compound
thereon, wherein the sample compounds on each of the protrusions
may be the same or different, and
a cantilever having a fixed end and a free end, the free end having
a substantially flat surface region having an area of at least 0.01
square microns and wherein the surface region is subdivided into a
plurality of spatially addressable subregions,
(b) chemically modifying the apical substrate region of each
protrusion to immobilize a sample compound thereon, wherein the
sample compounds on each of the protrusions may be the same or
different,
(c) chemically modifying the spatially addressable subregions of
the surface region of the free end of the cantilever to immobilize
a different reference compound on each subregion,
(d) coupling the sample support member to a sample stage portion of
an atomic force microscope and coupling the cantilever to a
cantilever-holding portion of the atomic force microscope, wherein
the atomic force microscope includes means for controlling the
relative position and orientation of the cantilever and the sample
support member to select a particular protrusion and to cause an
interaction between a reference compound immobilized on the surface
region of the free end of the cantilever and the sample compound
immobilized on the apical substrate area of the selected
protrusion, and means for measuring a physical parameter associated
with the interaction between the reference compound and the sample
compound,
(e) controlling the relative position and orientation of the
cantilever and the sample support member to select a particular
protrusion and to cause an interaction between a reference compound
immobilized on the selected subregion and the sample compound
immobilized on the selected protrusion, and
(f) measuring a physical parameter associated with the interaction
between the selected reference compound and the selected sample
compound.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (Prior art) is a schematic, enlarged representation of an
interaction between a conventional cantilever and a conventional
substrate.
FIG. 2 is a schematic, enlarged representation of an interaction
between a cantilever and a sample support member according to one
embodiment of the present invention.
FIG. 3 is a schematic, enlarged representation top view of a
portion of a sample support member according to one embodiment of
the present invention.
FIG. 4 is a schematic, enlarged side view of the free end of the
cantilever according to one embodiment of the present
invention.
FIG. 5 is a schematic, enlarged bottom view of the free end of the
cantilever according to another embodiment of the present
invention.
FIG. 6 is a schematic, enlarged top view of a sample support member
coupled to a force transducer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As shown schematically in FIG. 1 (prior art), a conventional atomic
force microscope cantilever 1 includes a sharp stylus or tip 2 that
may be modified to immobilize molecules 3 thereon. The stylus is
brought into contact with or proximity with a substrate 4 so that
an interaction between molecules on the stylus and molecules 5
immobilized on the substrate can be measured.
As shown schematically in FIG. 2, the apparatus of the present
invention preferably includes a cantilever 10 that has a free end
15 that has a surface region 20 that is chemically modified to
immobilize molecules 30 of a reference compound thereon. The sample
support member 40 includes a plurality of protrusions 45 each
having an apical substrate region 48 having sample molecules 50
immobilized thereon. According to the present invention,
interactions between reference molecules and sample molecules may
be measured by bringing the surface region of the cantilever into
contact with or into proximity with the apical substrate region of
one of the protrusions.
The sample support member includes a support base having a
plurality of protrusions that each have a chemically modified
apical substrate region. In general, the protrusions should be a
size and shape that allows only a small number of molecules of the
sample compound to be immobilized at the apex of each protrusion,
so that individual molecules of the sample compound can be isolated
for study. (Typically, the protrusions are visible only through an
optical microscope and cannot be seen clearly by the naked eye.)
Preferably, the protrusions are tapered or rounded with an apical
radius of curvature between about 5 and about 1000 nm. The apical
radius of curvature of a protrusion should be selected according to
the coverage (the number of molecules per unit area) of the
molecules of the sample compound to be immobilized thereon. For
compounds that are in relatively low abundance or coverage, or that
are difficult to immobilize, protrusions having relatively large
radii of curvature should be used; for example, rounded or
hemispherical shapes are preferred for these types of sample
compounds. The large radius of curvature helps to increase the odds
that every protrusion will have at least one sample molecule
immobilized thereon. For molecules that are easy to immobilize with
high coverage, tapered protrusions having relatively small radii of
curvature should be used. Pyramidal or conical shapes are preferred
for these types of sample compounds. The small radius of curvature
helps to assure that even though a large number of molecules may
become bound to each protrusion during the immobilization process,
single molecules at the apices of the protrusions can be isolated
for study. A sample support member can be constructed with a
variety of protrusions having differing radii of curvature, so that
the same support member can be used for immobilizing various types
of sample compounds.
In principle, there is no limit to the overall size of the sample
support member, although in practice, the size of the sample
support member will generally be selected according to the size of
the sample holding or stage area of the particular atomic force
microscope instrument being used. The size and spacing of the
protrusions may be selected to match the size of a particular
cantilever or reference compound support member so that the apical
substrate region of each protrusion is accessible to the cantilever
or reference compound support member. In particular, the
protrusions should be spaced apart at a distance greater than the
width of the reference compound support member so that the
reference compound support member may be maneuvered to address each
protrusion without interference from neighboring protrusions. On
the other hand, spacing the protrusions too far apart limits the
number of protrusions that can be packed into a small area.
Preferably, the width of the protrusions at their widest point is
about 0.5-5 .mu.m, and the distance between adjacent protrusions is
about 2-50 .mu.m. Thus, a sample support member that is a few
square centimeters in size may have millions of protrusions.
In some atomic force microscopes, a cantilever is held at a fixed
angle to the surface of the sample substrate while the cantilever
and the substrate are moved relative to each other. If this type of
microscope is used with a cantilever and sample support member of
the present invention, then the height of the protrusions can
affect how great of a surface area of the cantilever is accessible
to the apex of the protrusions. If a protrusion is of insufficient
height, the area of the cantilever that can be accessed in the
direction away from the free end is limited because the free end
would run into the sample support member. Preferably, the height of
the protrusions is about 1-10 .mu.m.
As shown schematically in FIG. 3, the sample support member 40 will
typically comprise a planar array of protrusions 45. Preferably,
the protrusions are arranged in a regular pattern, such as a square
array, so that it is easy to keep track of the location of any
particular protrusion. For convenience in keeping track of the
location of particular protrusions and distinguishing the
protrusions from each other, the sample support base may include
markings that are visible through an optical microscope. Also, for
convenience in distinguishing particular protrusions, groups of
protrusions may be separated by empty rows and columns.
Other configurations besides a planar array are possible. For
example, the sample support member could be a terraced structure
having at least one row of protrusions on each terrace.
The sample support member may be made of any material that can be
microfabricated. Preferably, the sample support member is a silicon
wafer. The protrusions are preferably integral with the support
base and may be made by standard microfabrication techniques, such
as photopatterning and etching of the sample support member. In
particular, methods of microfabrication similar to those used to
make conventional cantilever probe tips can be used to make the
protrusions of the sample support member. For example, a method of
making tapered microminiature silicon structures is described in
U.S. Pat. No. 5,201,992 and U.S. Pat. No. 5,204,581 to Andreadakis,
both incorporated herein by reference. The sample support member
may also be made by casting in a microfabricated mold. The sample
support member may be coated with one or more layers of material
that is useful for binding specific types of sample compounds. For
example, the sample support member may be coated with a layer of
gold, which allows the immobilization of thiol-containing
compounds.
The apical substrate regions of the protrusions are modified to
immobilize sample compounds thereon by any method known in the art
for covalently or non-covalently immobilizing a chemical or
biochemical entity on a substrate. In particular, chemical methods
used to modify conventional cantilever probe tips and sample
surfaces can be used to modify the protrusions of the present
invention. A general discussion of immobilization chemistry is
found in Lee et al, "Chemically-specific Probes for the Atomic
Force microscope", Israel Journal of Chemistry, Vol. 36, (1996), pp
81-87, incorporated herein by reference.
Electrochemical and electrostatic methods known in the art may also
be used to immobilize sample compounds on the protrusions. For
example, the electrostatic potential of the surface of the sample
support member may be manipulated to concentrate sample compounds
on the apices of the protrusions.
The sample compounds immobilized on the protrusions may be the same
or different, depending on the type of research that is being
conducted. For example, if a researcher is interested in studying
only the interaction of a reference compound on the cantilever with
only one specific sample compound, the sample support member can be
modified so that only one compound is immobilized on the
protrusions. On the other hand, if a researcher is interested in
studying the interaction of a reference compound with a number of
different sample compounds, the sample support member could be
modified so that different compounds are immobilized on different,
spatially addressable protrusions. Preferably, each sample compound
would be immobilized on a number of protrusions, so that if one
protrusion having a particular sample compound immobilized thereon
becomes damaged, another protrusion having the same sample compound
can be selected for study. Also, having each sample compound
immobilized on a number of different protrusions allows for
experiments to be repeated and for results to be double-checked
with statistical rigor. Any patterning method known in the art,
including, but not limited to, photo-patterning and .mu.-contact
printing, can be used to create arrays of protrusions having a
different compounds immobilized thereon. If electrochemical methods
are used to attach sample compounds to the protrusions, different
sample compounds may be patterned onto the sample support member by
using separate electrodes on different sections of the sample
support member. Moreover, methods of combinatorial synthesis can be
used to created combinatorial libraries in situ, with protrusions
having different members of the combinatorial library immobilized
thereon. Examples of methods of creating polypeptide or
oligonucleotide arrays are disclosed in Fodor et al,
"Light-Directed, Spatially Addressable Parallel Chemical
Synthesis", Science, 251 (1991), pp 767-773, U.S. Pat. No.
5,143,854 to Pirrung et al and U.S. Pat. No. 5,445,934 to Fodor et
al, the disclosures of which are incorporated herein by reference.
The methods described in these references can be adapted to
immobilize combinatorial libraries of sample compounds onto the
protrusions of the present invention.
In immobilizing a sample compound or sample compounds on the apical
substrate regions of the protrusions, it does not matter if a
particular method used has the incidental effect of also
immobilizing the sample compound on other portions of the sample
support member, as long as access to the sample compound
immobilized on the apical substrate region is not blocked.
By providing herein that the sample support member comprises a
sample support base having a plurality of protrusions wherein "each
protrusion" has an apical substrate region that is modified by the
immobilization of a sample compound thereon, it is not meant to
exclude from the scope of the invention instances wherein a sample
support member also has protrusions that are not chemically
modified. For example, a researcher may choose to chemically modify
only a portion of a sample support member and to leave the
remaining portion of the sample support member untouched. Or a
sample compound may be so difficult to immobilize onto a substrate
that only a fraction of the protrusions of a sample support member
are successfully modified and the remaining protrusions are
unmodified. These instances are within the scope of the
invention.
The present invention also includes a sample support member that is
modified to immobilize a linking compound on the apical substrate
regions of the protrusions, wherein the linking compound is a
compound that is capable of binding a sample compound. This
embodiment provides flexibility by allowing a researcher to select
a particular sample compound or sample compounds to bind to the
sample support member at a point in time subsequent to the initial
immobilization step. Any linking compound known in the art for
binding a sample compound to a substrate may be used. Typical
linking compounds are heterobifunctional crosslinkers that have a
portion of the molecule that is functionalized to bind a particular
type of substrate and a portion of the molecule that is
functionalized to bind particular type of sample compound. For
example, for binding the linking compound to a silicon substrate,
the linking compound may have one or more silane functional groups
of the formula --O(CH.sub.2).sub.n Si (OR).sub.3-m Cl.sub.m,
wherein R is CH.sub.3 or CH.sub.2 CH.sub.3, m is an integer from 0
to 3 and n is an integer from about 9 to about 25. For binding the
linking compound to a gold-layered substrate, the linking compound
may have one or more thiol functional groups. Likewise, the
sample-binding portion of the linking compound may be selected to
bind particular types of sample compounds such as proteins or
nucleic acids. For example, the linking compound may include one or
more maleimide groups that bind thiol-containing residues of
polypeptides or the linking compound may include one or more
succinimide groups that bind the amine groups of polypeptides or
nucleic acids. The linking compound may also be photoactivatable so
that the compound is capable of binding a sample compound only
after it has been irradiated. Examples of photoactivatable linking
compounds that can be immobilized on silicon are described in U.S.
application Ser. No. 08/797,325 to Conrad et al, filed Feb. 10,
1997, incorporated herein by reference.
It is expected that the sample support member of the present
invention will most commonly be used in atomic force microscopy
with a cantilever, as described herein. However, the sample support
member may also be used as a sample support for holding sample
compounds for other types of probe-based microscopy such as
scanning tunneling microscopy and scanning force microscopy, in any
set-up where it would be advantageous to have a sample immobilized
in a geometrically accessible fashion on the apices of a plurality
of protrusions. Other mechanisms for force transduction and other
types of platforms for holding a reference compound other than a
conventional cantilever may also be used. As used in this
application, the term "reference compound support member" refers to
any structure, whether a cantilever or not, that provides a surface
region on which the reference compound is immobilized so that it
can be brought into a position to interact with the sample compound
in a manner such that physical parameters associated with the
interaction can be measured. A mechanism for force transduction may
be coupled to the reference compound support member or the sample
support member. For example, in one variation of scanning probe
microscopy, described in Amrein, M. et al, "A Novel Force-sensing
Arrangement for Combined Scanning Force/Scanning Tunnelling
Microscopy Applied to Biological Objects" Journal of Microscopy,
Vol. 179, Pt. Sep. 3, 1995, pp 261-265, and in Schenk, M. et al,
"An Electret Microphone as a Force Sensor for Combined Scanning
Probe Microscopy", Ultramicroscopy 65 (1996), pp 109-118, both
incorporated herein by reference, a sample is mounted on top of a
force transducer which is an electret microphone membrane, and
deflections in the membrane in response to force exerted by a probe
tip are measured. The sample support member of the present
invention and reference compound support member could be adapted
for this type of arrangement or similar set-ups where the sample
support member is connected to a force transducer.
FIG. 6 is a schematic, enlarged top view of a portion of a sample
support member coupled to a force transducer. Preferably, the
reference compound support member is a cantilever. The cantilever
may be constructed of any material known in the art for use in
atomic force microscope cantilevers, including Si, SiO.sub.2,
Si.sub.3 N.sub.4, Si.sub.3 N.sub.4 O.sub.x, Al, or piezoelectric
materials. The chemical composition of the cantilever is not
critical and is preferably a material that can be easily
microfabricated and that has the requisite mechanical properties so
that it can be used for atomic force microscope measurements.
Likewise, the cantilever may be in any size and shape known in the
art for atomic force microscope cantilevers, except that, unlike
conventional cantilevers, it does not rely on having a stylus or
probe tip on its free end. Instead, the cantilever has a surface
region at the free end that is chemically modified by the
immobilization of at least one reference compound thereon.
Preferably, the cantilever is rectangular (a "diving board" shape)
or "V" shaped. The size of the cantilever preferably ranges from
about 5 microns to about 1000 microns in length, from about 1
micron to about 100 microns in width and from about 0.04 microns to
about 5 microns in thickness. Typical atomic force microscope
cantilevers are about 100 microns in length, about 20 microns in
width and about 0.3 microns in thickness. In general, increasing
the size of the cantilever allows for a larger chemically modified
surface region, allowing for a greater number of molecules to be
immobilized thereon. However, increasing the size of the cantilever
generally decreases the sensitivity of the cantilever to specific
intermolecular interactions and reduces the accuracy of force
measurements.
The fixed end of the cantilever may be adapted so that the
cantilever fits or interfaces with a cantilever-holding portion of
a conventional atomic force microscope.
The chemically modified surface region of the cantilever is on the
portion of the cantilever that, when the cantilever is incorporated
into an atomic force microscope, faces the sample support member.
The surface region is preferably a substantially flat area of at
least 0.01 square microns near the end of the cantilever.
The surface region of the free end of the cantilever may be
modified to immobilize a reference compound by any method known in
the art for immobilization of a chemical entity on a substrate.
Typically, the surface region will have billions or trillions of
molecules of the reference compound immobilized thereon.
In an alternative embodiment, the surface region of the cantilever
may be modified by immobilizing beads thereon. Beads of specified
sizes can be used to calibrate the radii of curvature or other
geometric attributes of the protrusions of the sample support
member. Also, beads can be used as a way of attaching a plurality
of different reference compounds to the cantilever. Different
reference compounds may be immobilized onto different sizes of
beads, that is, with each reference compound immobilized on a
specific size of bead, and the beads may then be immobilized
randomly onto the cantilever. In taking measurements of the
interaction of reference compounds with sample compounds
immobilized on a sample support member of the present invention,
particular reference compounds may be distinguished from each other
by the size of the bead to which they are attached. This embodiment
provides a convenient way to immobilize a plurality of different
reference compounds on the cantilever. Examples of bead sizes of
different categories that can be distinguished from each other are
1-2 nm beads, 10-15 nm beads and 20-25 nm beads. This embodiment is
shown in FIG. 4, which is a schematic, enlarged side view of the
free end of a cantilever 100 having small-sized beads 110 having a
first reference compound 115 immobilized thereon, medium-sized
beads 120 having a second reference compound 125 immobilized
thereon, and large beads 130 having a third reference compound 135
immobilized thereon.
In another alternative embodiment, the surface region of the
cantilever or reference compound support member may be subdivided
into spatially addressable subregions, with a different reference
compound immobilized on each subregion. The cantilever of this
embodiment may be created by any method known in the art for
creating spatially addressable arrays of immobilized compounds,
such as by photo-patterning. Although the number of spatially
addressable subregions is limited only by the resolution of
patterning techniques used to create the subregions and by the
resolution of whatever method is used to control the relative
position of the cantilever and the sample support member,
preferably the number of subregions is about ten or less. This
embodiment is shown in FIG. 5, which is a schematic, enlarged
bottom view of the free end of a cantilever 200 having subregions
210,220,230,240,250,260,270,280, and 290 having reference compounds
215, 225, 235, 245, 255, 265, 275, 285, and 295 immobilized
thereon.
By immobilizing as many as 10 different reference compounds on the
surface region of the reference compound support member and as many
as 1,000 different sample molecules on the protrusions of the
sample support member, it is possible to study as many as 10,000
different molecular interactions without changing the reference
compound support member or the sample support member. The methods
and apparatus of the present invention can be used to study or
screen receptor/ligand interactions, antibody/antigen interactions
and interactions of DNA or RNA with complimentary strands. For
example, a receptor can be immobilized on the surface region of the
reference compound support member and a large library of candidate
ligands can be screened to determine whether any of them interact
with the receptor and to measure the strength of the interaction.
With the present invention, rapid, efficient and statistically
rigorous large-scale screening of candidate entities for
pharmaceutical and agricultural utility becomes possible.
Alternatively, a ligand can be immobilized on the surface region of
the reference compound support member and a library of candidate
receptors may be screened to determine if the ligand interacts with
any of them. Similar methods can be used to screen antibody/antigen
interactions and the interactions of DNA or RNA with complimentary
strands.
To study molecular interactions between a reference compound and a
sample compound, the relative position and orientation of the
reference compound support member and the sample support member may
be controlled by any known means to select a particular protrusion
and to cause an interaction between the reference compound
immobilized on the surface region of the reference compound support
member and the sample compound immobilized on the apical substrate
area of the selected protrusion. Either the reference compound
support member or the sample support member or both can be moved,
scanned or oscillated. If the cantilever has different reference
compounds immobilized on different subregions, the relative
position of the cantilever and the sample substrate member may also
be controlled so that only a selected subregion of the cantilever
interacts with a selected protrusion. Preferably, the means for
controlling the relative position and orientation of the cantilever
and the sample support member is achieved through the use of
piezo-electric actuators, which can convert electrical signal into
mechanical displacements with sub-nanometer resolution.
Any physical parameter associated with the interaction between a
reference compound and a sample compound that can be measured by
atomic force microscopy can be measured by the apparatus and method
of the present invention. Physical parameters include, but are not
limited to, adhesion, elasticity, and changes in resonant
properties, such as amplitude, frequence, and phase. The physical
parameters may be measured by any known means including, but not
limited to, optical deflection, optical interferometry, and
piezoelectric effects. Preferably, forces of interaction between
the reference compound and the sample compound when they are
brought into or out of contact or into or out of proximity with
each other are measured by measuring deflections of the cantilever
from its equilibrium position. Deflections of the cantilever may be
measured by a number of techniques, including by reflecting a laser
beam off the back of the cantilever onto a position sensitive
detector.
Measurements may be carried out in any medium or environmental
conditions used in atomic force microscopy including, but not
limited to, under ambient conditions or under a liquid medium. In a
liquid medium, experimental conditions such as pH, ionic
concentration, and the presence of inhibitors or competitors can be
controlled and varied.
Commercially available atomic force microscope instruments may be
used for controlling the relative position of the cantilever and
the sample support member and for measuring physical parameters
associated with the interaction between reference compounds on the
cantilever and sample compounds on the sample support member.
Typically, commercial atomic force microscopes have removable
cantilevers and sample supports, so these instruments may be
modified by simply substituting the cantilevers and sample support
members of the present invention. The process of carrying out
measurements may be automated and computerized, so that a large
number of sample compounds, such as in a combinatorial library, can
be analyzed rapidly, efficiently and with statistical rigor. The
cantilever and sample support member of the present invention may
also be used with an atomic force microscope of a type that has
multiple, independently controlled cantilevers, such as is
described, for example, in U.S. Pat. No. 5,047,633 to Finlan et al,
incorporated herein by reference.
The apparatus of the present invention can also be used as a sensor
of environmental changes. In a sensor, compounds having a known
interaction are used as the reference compound and the sample
compound. A change in the interaction between the reference
compound and the sample compound indicates an environment change,
such as the presence of interfering molecules. The use of an atomic
force microscope as a sensor is described, for example, in U.S.
Pat. No. 5,372,930 to Colton et al.
Having described the invention, the following examples are given to
illustrate specific applications of the invention, including the
best mode now known to perform the invention. These specific
examples are not intended to limit the scope of the invention
described in this application.
EXAMPLE
A sample support member having a microfabricated array of tapered
protrusions was created by standard silicon microfabrication
techniques. Specifically, a silicon wafer was processed by forming
an oxide layer and then patterning the layer with a photoresist
pattern having systematically varying radii. The exposed oxide was
then etched away, leaving behind protected circular mask islands or
oxide caps. By anisotropic etching, the silicon layer was etched
around and underneath the oxide caps to create capped,
pyramid-shaped protrusions. The etching was continued until the
caps became detached. The remaining structures were touched up by
oxide formation and removal to produce an array of pyramid-shaped
protrusions with systematically varied apical radii of curvature.
Streptavidin was immobilized on the apices of the protrusions.
Biotinylated PEG and amino silane were immobilized on a Digital
Instruments NanoProbe silicon cantilever 20 .mu.m wide, 2 .mu.m
thick and 440 .mu.m long with a nominal force constant of 0.08 N/m.
The sample support member and the cantilever were fitted into a
modified commercial optical beam deflection based atomic force
microscope (Digital Instruments Nanoscope IIIa), which was used to
measure the forces of adhesion associated with the separation of
the cantilever and the sample support member. In this process, the
cantilever was centered over one of the pyramidal protrusions and
the z-piezo was excited with a triangle wave, resulting in a
relative vertical motion of 3 .mu.m at 10 Hz between the cantilever
and the protrusion. A stepper motor was used to lower the
cantilever into close proximity of the apex of the protrusion (less
than 2 .mu.m, but not touching). At this point, the z-piezo range
and rate were reduced to 500 nm and 1 Hz respectively. A triggering
algorithm was initiated so that the cantilever was inhibited from
applying more than 1.0 nN of load to the protrusion apex. Adhesion
measurements were taken and force curves were recorded. The
cantilever was then positioned over another protrusion and
measurements were repeated.
Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that, within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
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References